Anti-Inflammatory Effects of Paraprobiotic Lactiplantibacillus plantarum KU15122 in LPS-Induced RAW 264.7 Cells

Inflammation is a biodefense mechanism that provides protection against painful conditions such as inflammatory bowel disease, other gastrointestinal problems, and irritable bowel syndrome. Paraprobiotics have probiotic characteristics of intestinal modulation along with merits of safety and stability. In this study, heat-killed Lactiplantibacillus plantarum KU15122 (KU15122) was investigated for its anti-inflammatory properties. KU15122 was subjected to heat-killed treatment for enhancement of its safety, and its concentration was set at 8 log CFU/mL for conducting different experiments. Nitric oxide production was most remarkably reduced in the KU15122 group, whereas it was increased in the LPS-treated group. In RAW 264.7 cells, KU15122 inhibited the expression of inducible nitric oxide synthase, cyclooxygenase-2, interleukin (IL)-1β, IL-6, and tumor necrosis factor-α. ELISA revealed that among the tested strains, KU15122 exhibited the most significant reduction in PGE2, IL-1β, and IL-6. Moreover, KU15122 inhibited various factors involved in the nuclear factor-kappa B, activator protein-1, and mitogen-activated protein kinase pathways. In addition, KU15122 reduced the generation of reactive oxygen species. The anti-inflammatory effect of KU15122 was likely attributable to the bacterial exopolysaccharides. Conclusively, KU15122 exhibits anti-inflammatory potential against inflammatory diseases.


Introduction
Lactiplantibacillus plantarum stands as a significant species among the lactic acid bacteria (LAB) and possesses varied probiotic characteristics [1].L. plantarum is present in dairy products, fermented foods, and the mouth and intestinal tract of the host.L. plantarum is effective in treating various health conditions, including controlling the composition of fecal flora and preventing and treating irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), coronary heart disease, cancer, and other gastrointestinal problems [2].Non-viable microbial cells, often referred to as paraprobiotics, are safer and more stable than live probiotics.Heat-killed LAB can diminish the hazard of microbial infection and translocation of antibiotics resistance and are easier to store [3].
Reactive oxygen species (ROS) play a key role in multiple physiological functions and are triggered by LPS.Oxidative stress arises due to an disparity between the generation of free radicals and development of various biological conditions such as arthritis, chronic abdominal pain, cancer, and IBD [2].Individuals with IBS show decreased antioxidant capacity as a result of increased ROS, and alterations in the enzymatic system responsible for oxidative stress management may be involved in the development of IBS and its symptoms [10].
The objective of this study was to demonstrate the anti-inflammatory effect of L. plantarum KU15122 by inducing anti-inflammatory cytokines and suppressing proinflammatory cytokines and ROS in RAW 264.7 cells.In addition, involvement of the NF-κB, AP-1, and MAPK signaling pathways was confirmed.
Inflammation is a biodefense mechanism that provides protection against painful conditions such as inflammatory bowel disease, other gastrointestinal problems, and irritable bowel syndrome.Paraprobiotics have probiotic characteristics of intestinal modulation along with merits of safety and stability.In this study, heat-killed Lactiplantibacillus plantarum KU15122 (KU15122) was investigated for its anti-inflammatory properties.KU15122 was subjected to heat-killed treatment for enhancement of its safety, and its concentration was set at 8 log CFU/mL for conducting different experiments.Nitric oxide production was most remarkably reduced in the KU15122 group, whereas it was increased in the LPS-treated group.In RAW 264.7 cells, KU15122 inhibited the expression of inducible nitric oxide synthase, cyclooxygenase-2, interleukin (IL)-1β, IL-6, and tumor necrosis factor-α.ELISA revealed that among the tested strains, KU15122 exhibited the most significant reduction in PGE 2 , IL-1β, and IL-6.Moreover, KU15122 inhibited various factors involved in the nuclear factor-kappa B, activator protein-1, and mitogen-activated protein kinase pathways.In addition, KU15122 reduced the generation of reactive oxygen species.The anti-inflammatory effect of KU15122 was likely attributable to the bacterial exopolysaccharides.Conclusively, KU15122 exhibits anti-inflammatory potential against inflammatory diseases.

Signaling Pathway Analysis Using Western Blotting
RAW 264.7 cells were seeded in a 6-well plate (4 × 10 6 cells/well) overnight, and the samples were treated with LPS (1 μg/ml).Total protein was isolated from RAW 264.7 cells using lysis buffer (iNtRON Biotechnology, Republic of Korea) with a protease/phosphatase inhibitors.Twenty micrograms of each protein were fractionated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and moved onto a polyvinylidene fluoride (PVDF) membrane [15].Membranes were blocked with 5% skim milk in Tris-buffered saline with 1% Tween 20 (TBST) for 1 h, and were incubated with specific primary antibodies GAPDH (control), p38, p-p38, JNK, p-JNK, c-Jun, p-c-Jun, ERK, p-ERK, p65, p-p65, and IκB-α (Cell Signaling Technology Inc., USA) at 4°C for 16-24 h.After washing with TBST, the membranes were displayed to horseradish peroxidaseconjugated secondary antibodies (Cell Signaling Technology Inc.) for 1 h.Following a rinse with TBST, protein bands were identified using an improved chemiluminescence solution, and images were taken by displaying PVDF membranes to X-ray film.

ROS Production through Staining
RAW 264.7 cells (5 × 10 5 cells/well) were seeded into 12-well plates and cultured at 37°C [16].After 2 h, the cells were added samples and cultured with 1 μg/ml LPS for 18-24 h.Before removing the media, the wells were scrubbed twice with PBS.Each well was exposed with 20 μM 2,7-Dichlorodihydrofluorescein diacetate (DCFH-DA) (Sigma-Aldrich) and left undisturbed for 40 min in the darkroom.Images were captured using a DS-Ri2 digital camera (Nikon Co. Ltd., Japan) after the cells were observed under a fluorescence microscope (Nikon Co. Ltd.).

Production and Separation of Exopolysaccharides
The EPS obtained from each sample was purified using the ethanol precipitation [17].The bacterial suspension was centrifuged at 10,000 ×g for 10 min to acquire cell precipitates, which were then rinsed twice with 0.9% NaCl and centrifuged again.In the physical extraction method, cleaned bacterial pellets were reconstituted in 1 M NaCl, sonicated in a QSonica sonicator (USA) at 40 W for 3 min, and maintained on ice.Subsequently, supernatants of each treatment were acquired by centrifugation at 10,000 ×g for 10 min, then blended with twice their volume of ethanol and left to rest overnight at 4°C.The resulting EPS were collected after centrifugation at 10,000 ×g for 20 min and dissolved in ddH 2 O.The solution was stored at −80°C.
The dissolved EPS extract was evaluated using the phenol-sulfate method.A combination of EPS solution, 5% phenol, and sulfuric acid was prepared, and the presence of polysaccharides in the extract was indicated by an observable color reaction [18].

Statistical Analysis
Every experiments were examined in triplicate, and results are represented as the mean ± standard deviation.A difference of means was conducted using one-way analysis of variance (ANOVA), where significance was determined at p < 0.05.Statistical analyses were performed using SPSS software (version 18.0; SPSS Inc., USA).

Effects of Heat-Killed L. plantarum KU15122 on Cell Viability and NO Production
RAW 264.7 cells were used to access the effect of heat-killed L. plantarum KU15122 on cell viability to exclude that its anti-inflammatory properties are related to cytotoxicity.Heat-killed LGG, L. plantarum ATCC 14917, and L. plantarum KU15122 had no noticeable effects on cell viability at any of the concentrations tested (Fig. 1A).Proceeding with subsequent experiments using concentrations of heat-killed L. plantarum KU15122 that exhibited no cytotoxicity, ensuring no impact on its anti-inflammatory properties.
To assess the anti-inflammatory capacity of heat-killed L. plantarum KU15122, the effect of L. plantarum KU15122 on NO production in LPS-induced RAW 264.7 cells was examined.In the LPS-treated group, NO production was significantly higher compared to the LPS-negative group.In both the 8 log CFU/ml and 9 log CFU/ml, NO production were observed without cytotoxicity (data not shown).However, this study dealt on the overall mechanism at just 8 log CFU/ml.At 8 log CFU/ml, treatment with heat-killed L. plantarum KU15122 significantly inhibited NO production.Additionally, L. plantarum KU15122 exhibited lower NO production than that of LGG and L. plantarum ATCC 14917 (Fig. 1B).

Effect of Heat-Killed L. plantarum KU15122 on mRNA Expression of iNOS, COX-2, and Proinflammatory Cytokines
RT-PCR was performed to explore whether heat-killed L. plantarum KU15122 decreased the mRNA expression of iNOS, COX-2, and proinflammatory cytokines.In contrast to the negative group, LPS treatment resulted in notably elevated levels of iNOS, COX-2, IL-1β, IL-6, and TNF-α (Fig. 2A-2E).However, heat-killed L. plantarum KU15122 group showed the lowest levels of iNOS, COX-2, and proinflammatory cytokines.

Effect of Heat-Killed L. plantarum KU15122 on Protein Levels of PGE 2 , IL-1β, and IL-6
As per ELISA results, LPS activation notably prompted a significant rise in the transcriptional presence of PGE 2 , IL-1β, and IL-6.In contrast, heat-killed L. plantarum KU15122 treatment led to a reduction in the protein levels of PGE 2 , IL-1β, and IL-6 (Fig. 3A-3C).Compared with LGG and L. plantarum ATCC 14917, L. plantarum KU15122 showed similar or greater reduction in protein expression.

Effect of Heat-Killed L. plantarum KU15122 on NF-κB and AP-1 Signaling
To determine whether downregulation of proinflammatory factors was accompanied, the effect of heat-killed L. plantarum KU15122 on NF-κB and AP-1 signaling was assessed in LPS-induced RAW 264.7 cells.LPS stimulation led to marked phosphorylation of NF-κB such as IκB-α and p-p65.Compared with the positive

Effect of Heat-Killed L. plantarum on ROS Production in RAW 264.7 Cells
The impact of heat-killed L. plantarum KU15122 on the generation of ROS in RAW 264.7 cells induced by LPS was assessed.ROS production increased dramatically upon stimulation with LPS (positive control; Fig. 6B).Pretreatment with heat-killed LAB markedly reduced ROS production; moreover, L. plantarum KU15122 displayed an alleviating effect similar to that in the LPS-negative group (Fig. 6A-6F).

Bacterial EPS of L. plantarum KU15122 and Its Anti-Inflammatory Effect
L. plantarum KU15122 exhibited superior productivity compared with that of LGG and L. plantarum ATCC14917 (Fig. 7A).
MTT assays were conducted at different EPS concentrations (50, 100, 150, and 200 μg/ml), and no cytotoxicity was observed up to the maximum concentration of 200 μg/ml (data not shown).An NO assay using the extracted bacterial EPS showed results similar to those of paraprobiotics (Fig. 1).L. plantarum KU15122 had the best antiinflammatory effect, considering that the amount of NO produced was lower than that produced by LGG and L. plantarum ATCC 14197.From these findings, it can be inferred that the anti-inflammatory impact of L. plantarum KU15122 might be due to EPS (Fig. 7B).

Discussion
Extensive studies have been conducted on L. rhamnosus GG (LGG), a standard probiotic strain.L. plantarum ATCC 14917, recognized for its anti-inflammatory effects by suppressing inflammatory cytokines, has been suggested as a reference organism [2,19].Moreover, L. plantarum ATCC 14917 has shown beneficial effects in alleviating adipose inflammation [20] and preventing fatty liver disease [3].The functionality of L. plantarum KU15122 was assessed by comparing it with strains known for their excellent functionality.
NO is a labile radical and a ROS consisting of one nitrogen atom covalently bonded to a single oxygen atom with an unpaired electron.Proinflammatory cytokines induce the production of iNOS in monocytes, macrophages, neutrophils, granulocytes, and various other cells during inflammatory reactions [21].iNOS is induced in response to different agents, such as LPS or proinflammatory cytokines, through various signaling pathways [22].Major cellular receptors, such as Toll-like receptors and CD14, regulate and modulate iNOS activity in macrophages [23].Cell-free supernatant of L. plantarum WiKim0125 isolated from kimchi was decreased NO production and inflammatory cytokines, IL-1β, IL-6, and MCP-1 [24].PGE 2 serves various biological roles, including its active involvement in inflammation, where it facilitates local vasodilation, recruits, and activates inflammatory cells; it also act as an important marker of anti-inflammatory reactions, regulated by COX-2 [25].Additionally, PGE 2 has a significant impact on intestinal smooth muscle function in both healthy and diseased patients by causing contractions in small intestinal smooth muscle cells [26].According to a previous research, the levels of PGE 2 were found to correlate with the extent of inflammation and exhibited a repetitive pattern [26].Therefore, it was anticipated that L. plantarum KU15122 possesses potential anti-inflammatory activity through inhibition of these proinflammatory cytokines (Figs.2B and 3A).
The inflammatory cytokine TNF-α, alternatively referred to as cachectin, holds significance in certain pain models due to its pivotal role [28].IL-1β is released during infection, inflammation, and cell injury by monocytes and macrophages and by nonimmune cells as well [29].In addition, IL-6 signaling protein induces acute phase reactions in chronic diseases, typically those caused by immune stress [28].According to previous in vivo studies, L. plantarum 299v has shown effectiveness in decreasing the histological assessments and levels of cytokines linked to IBD across different animal studies involving colitis [30].Figs. 2 and 3 demonstrate that L. plantarum KU15122 effectively suppresses the generation of multiple inflammatory mediators and cytokines.
The primary regulatory transcription factor, NF-κB can form dimers, either by pairing with identical partners or with different ones such as p50 and p65 proteins.These dimers are initially held together by the inhibitor IkBα.The separation of these complexes is triggered by various factors, including cytokines, ultraviolet light, free radicals, stress, oxidized low-density lipoproteins, and bacterial and viral antigens [30].AP-1 is another major TLR-mediated transcription factor.Phosphorylated MAPK, particularly JNK, can also activate c-Jun [33].It was suggested that L. plantarum KU15122 reduces p-c-Jun and hampers the activation of the IKK-NF-κB signaling pathway, resulting in the formation of the AP-1 complex and a reduction in p65 nucleus entry in response to LPS stimulation.This lowers the production and release of inflammatory factors (Fig. 4A-4F).
Within the signaling network regulating cell growth and division, ERK, a member of the MAPK family, plays a crucial role.Inflammatory processes trigger the activation of the p38 and ERK signaling pathways, which have been shown to be critical in IL-6 production [33].JNK has a role in the development and function of T cells, as well as the production of proinflammatory cytokines like IL-2, IL-6, and TNF-α [34].Additionally, it was indicated that probiotics notably decreased the production of examined proinflammatory cytokines in cell culture, potentially by hindering the activation of the NF-κB and MAPK signaling pathways through TLR4 [35].The antiinflammatory activity of heat-killed L. plantarum KU15122 has been demonstrated by its ability to inhibit LPS receptors' expression of TLR-4-mediated MAPK signaling (Fig. 5A-5D).
Postbiotics like EPS, created by LAB, can engage with host cells as ligands, protecting the host by binding to pathogens in the gut [1].Similarly, EPS of L. plantarum has been extensively studied regarding its biological functions, structure, and genes [36].In addition to their use in pharmacology and nutraceuticals, EPS act as immunomodulatory, antimicrobial, antioxidant, cholesterol-lowering, anticancer, and prebiotic agents [37].Kwon et al. [38] demonstrated that EPS has anti-inflammatory effects.As expected, L. plantarum KU15122 yielded the highest amount of extracted EPS compared with that of LGG and L. plantarum ATCC 14917.Further, using the extracted EPS for NO production experiments, L. plantarum KU15122 exhibited a markedly inhibition rate, indicating significant suppression.While comparing the anti-inflammatory effects of bacterial samples and EPS, which is known for its anti-inflammatory properties, similar experimental outcomes were observed.Therefore, it can be inferred that the anti-inflammatory effect of heat-killed bacteria is attributable to EPS (Fig. 7).
In conclusion, this present study demonstrates that L. plantarum KU15122, isolated from traditional Korean kimchi, exhibits notable anti-inflammatory property.Specifically, heat-killed L. plantarum KU15122 effectively reduced the production of NO and proinflammatory cytokines in RAW 264.7 cells when activated by LPS.Furthermore, in LPS induced murine macrophages, the impact of heat-killed L. plantarum KU15122 was evaluated through its effects on the expression of proinflammatory mediators and cell signaling pathways such as NF-κB, AP-1, and MAPK pathways.The experimental results clearly indicated a significant reduction in inflammation following treatment with heat-killed L. plantarum KU15122, suggesting its potential effectiveness in mitigating conditions characterized by inflammation such as IBD and IBS.The study findings suggest the potential use of heat-killed L. plantarum KU15122 as a preventive agent against inflammation.

Fig. 1 .
Fig. 1.Effects of heat-killed LAB strains on cell viability and nitric oxide (NO) production in LPS-induced RAW 264.7 cells.(A) Cell viability, (B) NO production.NC, negative control without LPS; PC, positive control with LPS; LGG, L. rhamnosus GG; 14917, L. plantarum ATCC 14917; 15122, L. plantarum KU15122.Data are presented as mean ± standard deviation of triplicate experiments.Different letters on error bars represent significant differences (p < 0.05).

Fig. 7 .
Fig. 7. Total EPS production rate of LAB strains and its effect on NO production.(A) Total EPS production rate, (B) NO production.NC, negative control without LPS; PC, positive control with LPS; LGG, L. rhamnosus GG; 14917, L. plantarum ATCC 14917; 15122, L. plantarum KU15122.Data are presented as mean ± standard deviation of triplicate experiments.Different letters on error bars represent significant differences (p < 0.05).